RNA interference (RNAi) is a naturally occurring regulatory mechanism of most eukaryotic cells that uses small double stranded RNA (dsRNA) molecules to direct homology-dependent gene silencing. Its discovery by Fire and Mello in the worm C. elegans {Fire, 1998} was awarded the Nobel prize in 2006. Shortly after its first description, RNAi was also shown to occur in mammalian cells, not through long dsRNAs but by means of double-stranded small interfering RNAs (siRNAs) 21 nucleotides long {Elbashir, 2001}.
The process of RNA interference is thought to be an evolutionarily-conserved cellular defence mechanism used to prevent the expression of foreign genes and is commonly shared by diverse flora, where it is called post-transcriptional gene silencing, and phyla. Since the discovery of RNAi mechanism there has been an explosion of research to uncover new compounds that can selectively alter gene expression as a new way to treat human disease by addressing targets that are otherwise “undruggable” with traditional pharmaceutical approaches involving small molecules or proteins.
According to current knowledge, the mechanism of RNAi is initiated when long double stranded RNAs are processed by an RNase III-like protein known as Dicer. The protein Dicer typically contains an N-terminal RNA helicase domain, an RNA-binding so-called Piwi/Argonaute/Zwille (PAZ) domain, two RNase III domains and a double-stranded RNA binding domain (dsRBD) {Collins, 2005} and its activity leads to the processing of the long double stranded RNAs into 21-24 nucleotide double stranded siRNAs with 2 base 3′ overhangs and a 5′ phosphate and 3′ hydroxyl group. The resulting siRNA duplexes are then incorporated into the effector complex known as RNA-induced silencing complex (RISC), where the antisense or guide strand of the siRNA guides RISC to recognize and cleave target mRNA sequences {Elbashir, 2001} upon adenosine-triphosphate (ATP)-dependent unwinding of the double-stranded siRNA molecule through an RNA helicase activity {Nykanen, 2001}. The catalytic activity of RISC, which leads to mRNA degradation, is mediated by the endonuclease Argonaute 2 (AGO2) {Liu, 2004; Song, 2004}. AGO2 belongs to the highly conserved Argonaute family of proteins. Argonaute proteins are ˜100 KDa highly basic proteins that contain two common domains, namely PIWI and PAZ domains {Cerutti, 2000}. The PIWI domain is crucial for the interaction with Dicer and contains the nuclease activity responsible for the cleavage of mRNAs {Song, 2004}. AGO2 uses one strand of the siRNA duplex as a guide to find messenger RNAs containing complementary sequences and cleaves the phosphodiester backbone between bases 10 and 11 relative to the guide strand's 5′ end {Elbashir, 2001}. An important step during the activation of RISC is the cleavage of the sense or passenger strand by AGO2, removing this strand from the complex {Rand, 2005}. Crystallography studies analyzing the interaction between the siRNA guide strand and the PIWI domain reveal that it is only nucleotides 2 to 8 that constitute a “seed sequence” that directs target mRNA recognition by RISC {Ma, 2005}. Once the mRNA has been cleaved, and due to the presence of unprotected RNA ends in the fragments, the mRNA is further cleaved and degraded by intracellular nucleases and will no longer be translated into proteins {Orban, 2005} while RISC will be recycled for subsequent rounds {Hutvagner, 2002}. This constitutes a catalytic process leading to the selective reduction of specific mRNA molecules and the corresponding proteins. It is possible to exploit this native mechanism for gene silencing with the purpose of regulating any gene(s) of choice by directly delivering siRNAs effectors into the cells or tissues, where they will activate RISC and produce a potent and specific silencing of the targeted mRNA.
Many studies have been published describing the ideal features a siRNA should have to achieve maximum effectiveness, regarding length, structure, chemical composition, and sequence. Initial parameters for siRNA design were set out by Tuschl and co-workers in WO02/44321, although many subsequent studies and/or improvements have been published since then.
Also, a lot of effort has been put into enhancing siRNA stability as this is perceived as one of the main obstacles for therapy based on siRNA, given the ubiquitous nature of RNAses. One of the main strategies followed for stability enhancement has been the use of modified nucleotides such as 2′-O-methyl nucleotides, 2′-amino nucleotides, nucleotides containing 2′-O or 4′-C methylene bridges. Also, the modification of the ribonucleotide backbone connecting adjacent nucleotides has been described, mainly by the introduction of phosphorothioate modified nucleotides. It seems that enhanced stability is often inversely proportional to efficacy (Parish, 2000), and only a certain number, positions and/or combinations of modified nucleotides may result in a stable silencing compound. As this is an important hurdle within siRNA-based treatments, different studies have been published which describe certain modification patterns which show good results, examples of such are for example EP1527176, WO2008/050329, WO2008/104978 or WO2009/044392, although many more may be found in the literature.
Another strategy to achieve efficient siRNA delivery to target cells has been the use of lipids, which can envelope the siRNA compound, thus making it inaccessible to nucleases. As such, strategies based on siRNA packaging into liposomes have been described. Further sophisticated solutions along these lines are small nucleic acid lipid particles or SNALPs, which are described for example in patent applications US2006134189, US2006240093 or US2007135372. The lipids used may be cationic lipids, non-cationic lipids and conjugated lipids, even lipids containing polyalkylamine chains as a capturing agent of nucleic acid molecules have been used (WO2004/110499). Another alternative are the lipoplex formulations described in WO2007/121947, based on a liposome containing a helper lipid and a shielding compound which is bound to the nucleic acid, in which said shielding compound-nucleic acid complex is liberated from the lipid composition under in vivo conditions. In a specific embodiment the lipoplex formulation comprises a siRNA and a shielding compound which is a conjugate of PEG and ceramide.
The conjugation of lipid molecules to oligonucleotides such as cholesterol (Boutorine, 1993; Gryaznov, 1993; Zelphati, 1994) is shown to produce oligonucleotide conjugates with improved inhibitory properties (Godard, 1995; Le Doan, 1999; Soutscheck, 2004; Wolfrum, 2007). Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein targets siRNA primarily to the liver. As such different lipid conjugates will probably enhance delivery to different organs, thereby allowing treatment of different diseases.
In the present invention we describe the preparation and properties of oligonucleotides conjugated to sphingolipids.